Cellulose is one of the most abundant polymers found in nature and consists of glucose units connected by beta 1,4 linkages. The beta 1,4 linkages which connect individual glucose units are not easily degraded or depolymerized. However, there exists a variety of cellulase enzymes which are capable of enzymatically hydrolysing cellulose.
Cellulases are enzymes produced by a number of microorganisms which catalyse the hydrolysis of cellulose to products such as glucose, cellobiose, and other cellooligosaccharides. Cellulase is usually a generic term denoting a multienzyme mixture comprising exo-cellobiohydrolases (CBHs), endoglucanases (EGs) and β-glucosidases. Cellulase produced by the filamentous fingi Trichoderma longibrachiatum comprises at least two cellobiohydrolase enzymes termed CBHI and CBHII and at least 4 EG enzymes.
Cellulase enzymes work synergistically to hydrolzye cellulose to glucose. CBHI and CBHII generally act on the ends of the glucose polymers in cellulose microfibrils liberating cellobiose (Teeri and Koivula, 1995) while the endoglucanases act at random locations on the cellulose. Together these enzymes hydrolyse cellulose to smaller cello-oligosaccharides such as cellobiose. Cellobiose is hydrolysed to glucose by β-glucosidase. Both the exo-cellobiohydrolases and the endoglucanases are glycosyl hydrolases which hydrolyse the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non carbohydrate moiety.
The genes encoding CBHI, CBH II (Shoemaker et al., 1983; Teeri et al., 1987). EG I and EG II (Penttila et al., 1986; Saloheimo et al., 1988) have been cloned and isolated from filamentous fungi such as T. reesei and T. longibrachiatum. CBHI, CBH II and most EG proteins consist of a catalytic core domain and a cellulose binding domain (CBD) separated by a flexible linker region. The cellulose binding domain (CBD) promotes adsorption of the enzyme to regions of the cellulosic substrate (Tomme et al., 1988; Gilkes et al, 1992), while the core domain is responsible for catalysing the cleavage of cellulose. The linker region may ensure an optimal interdomain distance between the core domain and the cellulose binding domain (Teeri et al., 1992).
The major endoglucanases EG1, EG2 and EG3 are found at amounts of about 8 to 21% in Trichoderma relative to the total cellulase mixture consisting of CBH1, CBH2, EG1, EG2 and EG3 (Bisset, 1979; Hui et al., 2001). Hui et al., have determined EG1, EG2 and EG3 to be present in Trichoderma from 8 to 18% relative to the total cellulase mixture with EG3 present from 0 to 6% relative to the total cellulase mixture. Since Trichoderma EG3 lacks a cellulose binding domain (DNA sequence disclosed in U.S. Pat. No. 5,475,101), the natural abundance of endoglucanase core protein relative to the total endoglucanase mixture in Trichoderma is at most 33 wt % in the enzyme mixture. In Humicola insolens, the amount of endoglucanase protein relative to the total cellulase mixture is greater than 50% (Schulein et al.).
Several studies indicate that endoglucanase holo proteins are superior to endoglucanase core proteins for cellulose hydrolysis. EG2core protein from Trichoderma reesei does not bind as tightly to cellulose as EG2 (Macarron et. al., 1995; Nidetzky et. al., 1994) The EG2core protein is fully active against small soluble substrates such as the chromophoric glycosides derived from the cellodextrins and lactose. However, its activity against an insoluble cellulosic substrates such as Avicel (a crystalline type of cellulose) is greatly reduced compared to EG2 (Stahlberg et al., 1988; Nidetzcy et. al., 1994). Stahlberg showed that EG2 had seven fold more activity than EG2core. This was attributed to the fact that 79% of the EG2 adsorbed to the cellulose versus only 13% of the EG2core protein. Nidetzly examined the absorption and activities of EG2 and EG2core on filter paper. They disclose that on filter paper EG2 has four times more available sites than EG2core. Activity was found to depend on the extent of binding to the substrate. Kotiranta observed similar binding of EG2 and EG2core on steam pretreated willow, a lignocellulosic substrate. However the extent of cellulose conversion using EG2 or EG2core alone was extremely poor; using EG2core alone the extent of cellulose conversion was 1/2 that observed using EG2 (Kotiranta et al., 1999).
Schulein et al. disclose that by combining a Humicola Insolens exo-cellobiohydrolase CBHI and Humicola Insolens endoglucanasev in a molar ratio 90:5, a 55% conversion could be achieved in thirty hours. Substitution of endoglucanaseV core for endoglucanase V resulted in an almost 60% decrease in the rate of hydrolysis and the same conversion could only be achieved in 48 hrs.
The conversion of cellulose from cellulosic material into glucose is important in many industrial processes, such as the bioconversion of cellulose to fuel ethanol. Unfortunately, cellulose contained in most plant matter is not readily convertible to glucose, and this step represents a major hurdle in the commercialization of such a process. The efficient conversion of cellulose from cellulosic material into glucose was originally thought to involve liberating cellulose and hemicellulose from their complex with lignin. However, more recent processes focus on increasing the accessibility to cellulose within the lignocellulosic biomass followed by depolymerization of cellulose carbohydrate polymers to glucose. Increasing the accessibility to cellulose is most often accomplished by pretreating the cellulosic substrate.
The goal of most pretreatment methods is to deliver a sufficient combination of mechanical and chemical action so as to disrupt the fiber structure and improve the accessibility of the feedstock to cellulase enzymes. Mechanical action typically includes the use of pressure, grinding, milling, agitation, shredding, compression/expansion, or other types of mechanical action. Chemical action typically includes the use of heat (often steam), acid, and solvents. For example, one of the leading approaches to pretreatment is by steam explosion, using the process conditions described in U.S. Pat. No. 4,461,648 and also in Foody et al., 1980, both of which are incorporated herein by reference In this process, lignocellulosic biomass is loaded into a steam gun and up to 5% acid is optionally added to the biomass in the steam gun or in a presoak prior to loading the steam gun. The steam gun is then filled very quickly with steam and held at high pressure for a set length of cooking time. Once the cooking time elapses, the vessel is depressurized rapidly to expel the pretreated biomass.
Another approach described in U.S. Pat. No. 4,237,226, discloses the pretreatment of oak, newsprint, poplar, and corn stover by a continuous plug-flow reactor, a device that is similar to an extruder. Rotating screws convey a feedstock slurry through a small orifice, where mechanical and chemical action break down the fibers.
Pretreatment has been suggested to enhance delignification of the cellulosic substrate (Fan et al., 1981), create micropores by removing hemicellulose, change the crystallinity of the substrate, reduce the degree of polymerization of the cellulose (Knappert et al., 1980) and increase the surface area of the cellulosic substrate (Grethlein and Converse, 1991; Grohman et al., 1985).
Unfortunately, to date the approach of a pretreatment coupled with enzyme hydrolysis has not been able to produce glucose at a sufficiently low cost and make the conversion of cellulose to ethanol commercially attractive. Even with the most efficient of the current pretreatment processes, the amount of cellulase enzyme required to convert cellulose to glucose is high and this represents a significant cost in ethanol production. The option of adding less cellulase to the system usually decreases the amount of glucose produced to an unacceptable extent. The approach of decreasing the amount of enzyme required by increasing the length of time that the enzyme acts on the cellulose leads to uneconomical process productivity, stemming from the high cost associated with retailing the enzymatic mixtures in hydrolysis tanks.
Thus there is a need within the art to identify new methods that enhance the conversion of cellulose within a cellulosic substrate to glucose. Further there is a need in the art to identify enzymes or mixtures of enzymes which enhance the conversion of cellulose to glucose and which are recoverable, recyclable, and reusable.
It is an object of the present invention to overcome drawback of the prior art.
The above object is met by a combination of the features of the main claims. The sub claims disclose further advantageous embodiments of the invention.